The dorsal tegmental acetylcholinesterase (AChE) containing pathway was lesioned in the midbrain reticular formation. The lesions involved AChE positive perikarya in the n. cuneiformis. A four day survival period was allowed before examination of the brain for histochemical changes in AChE and biochemical changes in choline acetyltransferase (CAT). A decrease in AChE staining and CAT activity was observed in the ipsilateral anterior thalamic nuclei, n. reticularis thalami, n. medialis thalami, n. parafascicularis, the pretectal nuclei, the lateral geniculate and the superior colliculus. CAT, unlike AChE, is a specific marker for cholinergic neurons. In the areas with decreased staining for AChE, CAT activity was also decreased to 40-60 percent of control values obtained from sham rats. This study provides evidence that a major cholinergic pathway emanates from the region of the n. cuneiformis. The nucleus of the tractus diagonalis was found to contain a major cholinergic pathway that projects via the stria medullaris, habenula, and fasciculus retroflexus to provide 50 percent of the cholinergic innervation to the interpeduncular nucleus.